Bioactive Materials 5 (2020) 82–91

Contents lists available at ScienceDirect

Bioactive Materials
journal homepage: http://www.keaipublishing.com/biomat

3D printing of bone tissue engineering scaffolds T

a,∗,1 b,1 e,1 a a a a
Chong Wang , Wei Huang , Yu Zhou , Libing He , Zhi He , Ziling Chen , Xiao He ,
Shuo Tianb, Jiaming Liaoa, Bingheng Lua, Yen Weid, Min Wangc,∗∗
a
School of Mechanical Engineering, Dongguan University of Technology, Songshan Lake, Dongguan, Guangdong, PR China
b
Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, PR China
c
Department of Mechanical Engineering, The University of Hong Kong, Pokfulam Road, Hong Kong, Hong Kong SAR, PR China
d
Department of Chemistry, Tsinghua University, Beijing, PR China
e
Institute of Biomedical and health engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong, PR China

A R T I C LE I N FO A B S T R A C T

Keywords: Tissue engineering is promising in realizing successful treatments of human body tissue loss that current
3D printing methods cannot treat well or achieve satisfactory clinical outcomes. In scaffold-based bone tissue engineering, a
Bone tissue engineering high performance scaffold underpins the success of a bone tissue engineering strategy and a major direction in
Scaffold the field is to produce bone tissue engineering scaffolds with desirable shape, structural, physical, chemical and
Bioceramic
biological features for enhanced biological performance and for regenerating complex bone tissues. Three-di-
Polyester
Hydrogel
mensional (3D) printing can produce customized scaffolds that are highly desirable for bone tissue engineering.
Biomolecule The enormous interest in 3D printing and 3D printed objects by the science, engineering and medical com-
Controlled release munities has led to various developments of the 3D printing technology and wide investigations of 3D printed
products in many industries, including biomedical engineering, over the past decade. It is now possible to create
novel bone tissue engineering scaffolds with customized shape, architecture, favorable macro-micro structure,
wettability, mechanical strength and cellular responses. This article provides a concise review of recent advances
in the R & D of 3D printing of bone tissue engineering scaffolds. It also presents our philosophy and research in
the designing and fabrication of bone tissue engineering scaffolds through 3D printing.

1. Introduction ink jet 3D printing, adhesive droplet and powder bed-based AM, digital
laser processing, continuous liquid interface production, etc., have been
Three-dimensional (3D) printing, also known as additive manu- developed based on different working principles [13–18]. The 3D
facturing (AM) and rapid prototyping, is a process of joining materials printed materials and products are advantageous in following aspects:
to make objects from 3D model data, usually layer-by-layer, as opposed customized shape, tailored pore size/porosity, tuned mechanical
to subtractive manufacturing methodologies [1]. 3D printing is a ver- properties, etc. The number of publications on 3D printing, its appli-
satile technique to fabricate a variety types of materials including cation in different industries and 3D printing products has sky-rocketed
polymers, ceramics, metals and composites, with customized shapes over the past decades and in 2018–2019 alone there were about 15,726
and dense or macro/micro porous architecture [2–7]. 3D printed ob- journal publications on 3D printing and associated topics.
jects can be used in many industries for applications such as manu- 3D printing has attracted great attention in the biomedical field for
facturing of turbine blade, jewelry designing, mould making, building, two main reasons: the technology itself - its versatility, ease of use and
tissue engineering, etc [8–12]. The origin of 3D printing dates back to precise control of the fabrication process, and the products it produces -
late 19th century, when photosculpture and geomorphology technolo- customized shape and structure possessing unique architecture and
gies were developed. Between 1980s and 2010s, a number of 3D properties that are highly sought after for biomedical applications. In
printing techniques including fused deposition modeling (FDM), se- the biomedical field, the number of peer-reviewed articles on 3D
lective laser sintering (SLS), stereolithography, selective laser melting, printing, 3D printed structures and their potential clinical applications

Peer review under responsibility of KeAi Communications Co., Ltd.

∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: [email protected] (C. Wang), [email protected] (M. Wang).
1
Chong Wang, Wei Huang and Yu Zhou contributed equally.

https://doi.org/10.1016/j.bioactmat.2020.01.004
Received 18 July 2019; Received in revised form 15 December 2019; Accepted 7 January 2020
Available online 15 January 2020
2452-199X/ © 2020 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
C. Wang, et al. Bioactive Materials 5 (2020) 82–91

has increased exponentially from 8 publications in 2002–2906 pub- blended with biodegradable synthetic/natural polymers to make 3D
lications in 2018. Good review articles on 3D printing and associated scaffolds through micro-extrusion without post-sintering. The use of
topics, which are relevant to biomedical engineering, have been written composites as printing inks not only provides scaffolds with osteo-
by leading or established researchers [19–23] and the 3D printing conductivity but also improves the degradability of the inorganic par-
technology and 3D printing products have been constantly investigated ticulate fillers and alters physical, mechanical and biological properties
worldwide by an increasing number of researchers, established or new of scaffolds. In addition, functional agents such as drugs, biomolecules
in the biomedical field. For human bone tissue regeneration, a few could also be incorporated to provide scaffolds with capabilities of anti-
strategies hold great promises. Among these strategies, scaffold-based bacteria, osteogenesis, angiogenesis, etc.
tissue engineering has been widely and intensively investigated [24]. A
tissue engineering scaffold should provide a conductive microenviron- 2.2. Structure
ment for the selected cells and recent trend is to create customized
scaffolds with pre-designed shapes, structure and functions for en- Appropriate macro- and microstructures are key features in bone
hanced tissue regeneration [25]. Among numerous methods, 3D tissue engineering scaffolds. On one hand, patterned macropores in
printing has been considered as an advantageous technique in fabri- scaffolds could influence the cell penetration and cell distribution, and
cating tissue engineering scaffolds, as the 3D printed macro-micro most importantly, enables the transportation of gases and nutrients into
structure could morphologically mimic the multi-scale structure of the deeper layer of scaffolds, hence maintaining the cell viability at a
human body tissues [26]. Apart from this, scaffolds produced through high level [35]. On the other hand, micropores on scaffold struts are
certain 3D printing technique are excellent delivery vehicles to provide crucial in determining the initial cellular responses such as cell at-
local, sustained release of drugs and/or biomolecules [27]. Many in- tachment and cell spreading [36]. Moreover, adjusting the size of mi-
vestigations have been and are being conducted to impart 3D printed cropores could tune the release behaviour of drugs/biomolecules/
scaffolds with new functions [28]. By appropriate surface modification, agents loaded in the scaffold matrix or adsorbed/absorbed/conjugated
biologically active molecules and cell recognizable ligands can be on the scaffold surface.
physically or chemically linked to scaffold surface. Functional nano-
particles and drugs can also be directly mixed with a synthetic/natural 2.3. Mechanical properties
polymer solution which is then 3D printed to form functional tissue
engineering scaffolds. This article provides a concise review of recent Human bone tissue comprise of cortical bone and cancellous bone.
advances in the R & D of 3D printing of bone tissue engineering scaf- Cortical bone is dense, showing a porosity of only 5–10%, while can-
folds. It also gives our philosophy in designing and making advanced cellous bone, which has a spongy-like structure, has a porosity of
bone tissue engineering scaffolds with appropriate functions and pre- 50–90%. Cortical bone accounts for up to 80% of the weight of human
sents some of our recent research in this area. skeleton while cancellous bone accounts for the rest 20%. Cortical bone
has significantly higher compressive strength and Young's modulus
2. Requirements of 3D printed bone tissue engineering scaffolds than cancellous bone [37]. Therefore, when repairing load bearing and
non-load bearing bone tissues, the 3D printed bone tissue engineering
The regeneration of bone is complex because it involves a number of scaffolds should have match mechanical properties in order to provide
molecular, cellular, biochemical and mechanical cues. Therefore, sufficient mechanical support and avoid stress shielding.
porous bone tissue engineering scaffolds with appropriate shape, pore
size, porosity, degradability, biocompatibility, mechanical properties 2.4. Cellular responses
and desirable cellular responses are required to induce bone regenera-
tion. Whether 3D printed bone tissue engineering scaffolds could induce
favorable cellular responses is also of great importance in scaffold de-
2.1. Composition signing. First and foremost, the scaffold should be biocompatible,
showing no acute or long-term toxicity in vitro and in vivo. Secondly, as
Bioceramic powders, natural/synthetic hydrogels, non-hydrogel- the initial cell attachment, spreading and proliferation closely relate to
based polymers and their composites have been adopted as raw mate- the scaffold surface properties such as wettability and surface chem-
rials to formulate printing inks for 3D printing. Biodegradable and istry, providing scaffolds with suitable surface hydrophilicity is desir-
biocompatible polyesters such as poly(L-lactic acid) (PLLA), poly(vinyl able and this can be achieved by coating hydrophilic polymers and bio-
alcohol) (PVA), poly-β-hydroxybutyrate (PHB), polyurethane elasto- agents such as peptides and biomolecules on scaffold surface. Besides,
mers, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(D,L- to improve bone regeneration, the controlled delivery of osteoconduc-
lactic acid) (PDLLA), poly(lactic-co-lgycolic acid) (PLGA) and poly- tive and osteoinductive agents in scaffolds is strongly recommended to
caprolactone (PCL) and polyurethanes can be processed into wires, induce sufficient osteogenesis in vitro and in vivo. Last but not the least,
pellets and even powders to enable 3D printing of polymer scaffolds growth factors to enhance vascular formation and angiogenesis can also
with the assistance of high temperature melting-extrusion and sin- be delivered to improve the scaffold vascularization during bone re-
tering, or dissolved in organic/aqueous solvents to allow micro extru- generation process.
sion-based 3D printing at room/low temperature [29–34]. Likewise,
hydrogel precursors made of aqueous solutions of natural polymers 3. Ceramic-based bone tissue engineering scaffolds
including chitosan, collagen, gelatin, gelatin methacryloyl, hyaluronic
acid, sodium alginate and polyethelyene glycol diacrylate (PEGDA) 3.1. 3D printing of ceramic scaffolds followed by sintering
could be 3D printed into scaffolds at room- or low temperature and
further stabilized via UV-, ion- or temperature-assisted crosslinking. To date, 3D printed bioceramic scaffolds have been widely used as
Bioceramic precursors or bioceramic powders including micro- or na- bone tissue engineering scaffolds, as bioceramic scaffolds are me-
nosized calcium phosphates (Ca–P), hydroxyapatite (HA), β-tricalcium chanically, structurally and compositionally similar to bone apatite in
phosphate (β-TCP), carbonated calcium deficient hydroxyapatite native bone tissue. The most employed strategy to produce ceramic-
(CDHA), bioactive glasses, etc., can be blended with binders or photo- based bone scaffolds is to print “green body” scaffolds with customized
polymers to form printing inks to make 3D printed “green body”, which shape and pore size/porosity, followed by high temperature sintering,
are further sintered to remove the organic phase, leaving only hard which burns out all organic phases, forming pure ceramic scaffolds. Size
bioceramic scaffolds. Bioceramic micro/nanoparticles can be further shrinkage may occur after sintering but the mechanical strength and

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Table 1
Comparison of bone tissue engineering scaffolds made through different 3D printing techniques.
Technique Illustrative schematic Advantage Challenge Ref

3D printing of ceramic based Scaffolds are mechanically, Unsuitable for load bearing [38–40], [42–44]
bone tissue engineering structurally and compositionally applications; relatively low
scaffolds similar to native bone; scaffolds have compressive strength and
higher porosity, controlled swelling modulus; brittleness
profile, enhanced biomineralization
capacity and osteogenic property;
polymer coating on scaffolds promotes
bone ingrowth with improved
osteoblast cell viability and
proliferation under hypoxic
conditions,
3D printing of hydrogel based High water content; relatively high PVA resist protein absorption and [45–53]
bone tissue engineering tensile strength; large stretchability; cell adhesion; much lowered
scaffolds excellent protein/cell loading ability; compressive strength than that of
controlled release of biomolecules/ the human cancellous bone tissue;
drugs; micro/nanoporous structure for quick degradation
up-regulated cell attachment,
proliferation and osteogenic
differentiation.

3D printing of polyester Simplified operation process; greater Uneven distribution of bioceramic [54–62,70–78]
scaffolds at high convenience and flexibility; excellent particles; defects between
temperature & post- reproducibility; eco-friendly; high bioceramics and polyester due to
treatment printing resolution; better cell bad interaction; scaffolds with a
colonization and proliferation; higher porosity have lower
incorporate bioceramic particles to mechanical strength; quick release
improve mechanical properties and of biomolecules
wettability; coating of natural
polymers on scaffold surface improves
cell attachment; delivery of
biomolecules improve osteogenesis
and angiogenesis
Cryogenic 3D printing of bone Excellent compressive strength; in situ – [63–65]
tissue engineering delivery of biomolecules with
scaffolds preserved biological activity; good
biodegradability and biocompatibility;
macro/micro porous structure for
improved cell adhesion and
infiltration; controlled release of
biomolecules; incorporation of
bioceramics for better osteogenesis;
incorproation of graphene oxide
enhances the osteogenesis

3D printing of synthetic/ Electrospun fibers can be printed into Limited by the maximum number [66–69,80–83]
natural polymer bone regular pattern with varied layers; of layers that can be produced
tissue engineering incorporation of nHA in polyesters before control over fibre
scaffolds via electrostatic provides scaffolds with good placement is lost due to the
field-assisted micro biocompatibility and facilitated accumulation of instabilities
extrusion cellular alignment and proliferation in
vitro.

Young's modulus could be greatly increased. Seidenstuecker et al. mechanically strong and supported both osteogenic differentiation of
produced bioglass/β-TCP green body scaffolds via micro extrusion- mesenchymal stem cells (MSCs) and chondrogenic differentiation of
based 3D printing at room temperature, using dextrin as binder to bind chondrocytes in vitro and in vivo. The advantages and disadvantages of
bioglass and β-TCP powders, followed by high temperature sintering scaffolds made through major 3D printing techniques are listed in
[38]. Although the mechanical strengths of the scaffolds were Table 1.
0.17–0.64 MPa, making them unsuitable for load bearing applications,
these scaffolds can be used as bone fillers due to the improved cellular
3.2. 3D printing of ceramic scaffolds at room/low temperature
responses. Song et al. used both cryogenic 3D printing and post-sin-
tering to produce bone tissue engineering scaffolds with both hier-
Apart from high temperature sintering, in recent years, an in-
archically porous structure (interconnective macro pores and micro-
creasing number of studies adopted non-sintered ceramic scaffolds to
holes on strut surface) and superior compressive strength [39]. Chen
induce bone tissue regeneration. On this condition, a small portion of
et al. constructed a lithium-calcium-silicate crystal bioscaffold with
organic/natural polymer binders are used to bind ceramic powders and
dual bioactivities for osteochondral interface reconstruction through 3D
not removed after 3D printing. Song et al. reported the fabrication of
printing, followed by post-sintering [40]. The scaffolds were
nano-biphasic calcium phosphate (nBCP)/PVA (mass ratio of nBCP:

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PVA = 84: 16) composites incorporated with platelet-rich fibrin using could be used for bone tissue engineering. Lee et al. fabricated micro/
an extrusion-based low temperature 3D printing [41]. Improved in vitro nanoporous collagen/decellularized extracellular matrix (ECM)/silk-fi-
biocompatibility and biological activity toward bone marrow-derived broin biocomposite scaffolds using a low temperature 3D printing [47].
MSCs (BMSCs) such as BMSC adhesion, proliferation, and osteogenic The micro/nanoporous structure and the ECM-like composition were
differentiation were obtained and a greater extent of appropriate bone favorable for inducing up-regulated cell attachment, proliferation and
formation in a critical-size segmental bone defect model in rabbits was osteogenic differentiation in vitro. However, the compressive strength
also observed. However, the scaffolds had a relatively low compressive and modulus were significantly lower than that of human cancellous
strength and modulus, showing only 1/10 of that of human cancellous bone, making them a bit difficult to fill the requirements for bone tissue
bone. To mimic the natural architecture of hollow bones, which allow repair. Gao et al. produced hydrogel scaffolds with gradient composi-
nutrient exchange and bone neovascularization, Boga et al. fabricated tion/structure and excellent mechanical strength through direct 3D
cylindrical graphene oxide/TCP/alginic acid (GO/TCP/AA) scaffolds printing [48]. The cartilage layer and the subchondral layer in the
using a Fab@home 3D printer [42]. The GO containing scaffolds gradient scaffolds had distinct microscopic structures, compositions and
showed higher porosity, without impairing their mechanical properties. anisotropic mechanical strengths. The gradient scaffolds could induce in
These scaffolds also presented a controlled swelling profile, enhanced vitro chondrogenic and osteogenic differentiation of MSCs in a spatial-
biomineralization capacity and increased alkaline phosphatase (ALP) tempro manner. Moreover, the in vivo study also confirmed their ef-
activity. fectiveness in simultaneously achieving new bone formation and car-
tilage repair, which is highly desirable for the osteochondral tissue
3.3. Post-functionalization of 3D printed ceramic scaffolds regeneration. Luo et al. used concentrated alginate/gelatin solutions as
printing inks to make scaffolds via direct 3D printing, followed by
To further provide bioceramic scaffolds with additional functions, homogeneous precipitation of nanoapatite coating on scaffold surface
post-functionalization including coating and post-adsorption of func- [49]. The biomimetic scaffolds had excellent mechanical properties
tional agents has been widely adopted. Touri et al. fabricated latticed which were higher than that of human cancellous bone and had ex-
scaffolds from the robocasting paste containing 60 vol% HA and 40 vol cellent capability of adsorbing proteins. The precipitated apatite sti-
% β-TCP through 3D printing, followed by sintering [43]. The sintered mulated the proliferation and osteogenic differentiation of rat BMSCs
3D scaffolds were further coated with calcium peroxide (CPO)/PCL on scaffolds. They also produced 3D bioprinted scaffolds using alginate/
composite through dip-coating for in situ production of oxygen in the PVA as inks [50]. By adjusting the mass ratio between alginate and
implanted sites. The oxygen released in a sustained manner and was PVA, scaffolds without collapse can be constructed, followed by im-
dependant on the concentration of CPO encapsulated in the PCL coating mersion in CaCl2 solution for fully crosslinking. Moreover, bovine
matrix. The coated scaffolds could provide a great potential for pro- serum albumin (BSA) and recombinant human BMP-2 (rhBMP-2) were
moting bone ingrowth with improved osteoblast cell viability and encapsulated in alginate hydrogel microparticles and loaded in printing
proliferation under hypoxic conditions. Kim et al. incorporated bone inks to produce BSA or rhBMP-2 loaded alginate/PVA scaffolds, in
morphogenetic protein-2 (BMP-2)-loaded PLGA nanoparticles on the which the release behaviour of BSA and rhBMP-2 can be controlled by
surface of a 3D printed HA scaffold using an PCL emulsion coating adjusting the pore size of microparticles. Heo et al. incorporated bone
method [44]. BMP-2/PLGA nanoparticles were uniformly distributed forming peptide into alginate through EDC/NHS coupling to formulate
on the scaffold surface and BMP-2 was released gradually. Moreover, bioinks and printed them into hybrid bone tissue engineering scaffolds
PCL coating improved the compressive strength of the scaffold. The in [51]. In vitro and in vivo results demonstrated that the alginate-based
vitro cell proliferation, adhesion, and osteogenic differentiation and in scaffolds provided a stable environment for the growth of human adi-
vivo new bone formation were improved with PCL-BMP-2/PLGA na- pose-derived stem cells and induced synergistic effect to enhance bone
noparticle coated scaffold. regeneration. However, the mechanical properties of the scaffolds were
not studied.
4. Hydrogel bone tissue engineering scaffolds

4.1. 3D printing of hydrogel scaffolds 4.2. 3D printing of cell-laden hydrogel scaffolds

In recent years, due to the capability of encapsulating biomolecules Zhai et al. 3D printed osteoblast-laden nanocomposite constructs
and cells in situ, hydrogels have been used to make bone tissue en- based on PEGDA/nanoclay/hyaluronic acid sodium salt hydrogel bio-
gineering scaffolds via extrusion-based 3D printing. By tuning the hy- inks, with a two-channel 3D bioprinting method [52]. The cell-laden
drogel composition, scaffolds with varied mechanical properties and PEG/clay constructs not only encapsulated osteoblasts with higher than
cellular responses can be obtained, and some of them showed potential 95% viability in the short-term but also exhibited excellent osteogenic
in bone regeneration. Kim et al. employed gelatin/PVA solution as ability in the long-term due to the release of bioactive ions such as
printing inks to produce tissue engineering scaffolds for hard tissue magnesium ions and silicon ions, which induced the suitable micro-
repair on a low temperature plate [45]. The addition of PVA was to environment to promote the differentiation of the loaded exogenous
provide biocompatible gelatin scaffolds with improved mechanical cells in vitro and in vivo, suggesting a promising way for bone tissue
strength. Through composition optimization, scaffolds with excellent regeneration in terms of cell engraftment, survival, and ultimately long-
printability and porous structure can be produced. The gelatin/PVA term function. Demirtas et al. reported MC3T3-E1 pre-osteoblastic cell-
scaffolds with a gelatin: PVA ratio of 1:1 showed both improved me- laden chitosan and chitosan-HA hydrogels were produced via extrusion-
chanical properties and enhanced cell activities such as improved cell based 3D printing [53]. Cells printed within chitosan/HA composite
seeding efficiency, proliferation and osteogenic differentiation. How- hydrogel had peak expression levels for early- and late stage osteogenic
ever, the compressive strength was still much lower than that of the markers. Compared to alginate-based hydrogels, chitosan-based hy-
human cancellous bone tissue. Sithole et al. produced a polymeric drogels showed superior printability and potential to act as suitable bio-
scaffold by employing sodium alginate as a bio-ink which interacted inks for making cell-laden hydrogel scaffolds for bone tissue en-
with a poly (ethyleneimine) solution during 3D bioprinting to form a gineering. However, the elastic modulus (15 kPa) is significantly lower
polyelectrolyte complex through ionic bond formation. The silica gel than that of human cancellous bone.
was included in the bio-ink as temporal inorganic support component
and for ultimate enhancement of osteoinduction [46]. The fabricated
scaffolds had relatively high tensile strength, suggesting this scaffold

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5. Non-hydrogel-based polymer bone tissue engineering scaffolds significantly up-regulated on solvent-treated scaffolds. Other methods
are also developed to improve the cell attachment by increasing the
5.1. 3D printing of polyester scaffolds at high temperature wettability of scaffolds produced through FDM. Park et al. fabricated
PCL/PEG scaffolds through FDM, followed by washing out of water
Within the employment of extrusion-based 3D printing, apart from soluble PEG to form micropores on thread surface [59]. The wettability
ceramics and hydrogels, polyesters can be used as the main component of PCL scaffolds was significantly improved and improved cell pro-
of printing inks to make bone tissue engineering scaffolds. Due to the liferation was also observed on PCL/PEG scaffolds. These findings in-
advantages such as simplified and eco-friendly operation process and dicate that 3D printing of scaffolds with partially sacrificial features is a
low cost, FDM which uses wires or pellets made from PLA, PLGA, PCL promising way to endow scaffold with advanced cellular responses. In
and other biodegradable and biocompatible polyesters are often addition to coating hydrophilic molecules on thread surface of scaf-
adopted to produce bone tissue engineering scaffolds. Upon continuous folds, many biomolecules which could induce desirable cellular re-
transport of raw materials into the heated chamber connected with a sponses have also been coated on the scaffold surface. Teixeira et al.
metal nozzle, polyester wires and pellets are melted and extruded from coated mussel-inspired polydopamine (PDA) and type I collagen onto
the nozzle to draw pre-designed pattern in a programmed way, fol- the surface of PLA scaffolds fabricated through FDM by directly im-
lowing the CAD model. The exuded microfluidic patterns solidify mersing PLA scaffolds into dopamine solution for 24 h within a shaking
quickly due to the heat dissipation at the room temperature, hence water bath [60]. The type I collagen solution was then conjugated to
enabling the layer-by-layer building of the polyester scaffolds for bone the PDA-coated PLA scaffolds with the assistance of EDC/NHS for 48 h.
tissue engineering. Gremare et al. produced PLLA scaffolds via FDM and The coating of PDA increased the coupling amount of type I collagen on
evaluated the physical, chemical and biological performance. The pore the scaffolds and cells showed the highest cell density on the PLA
size (0–250 μm) was identical to that in the CAD file, whereas the scaffolds coated with PDA/collagen and showed significantly up-regu-
thread diameter (120 μm) was smaller than that designed in CAD file lated expression level of vinculin adhesive plaques and F-actin cytos-
[54]. Similar tensile strength was obtained for different scaffolds and all keleton. In addition, greatly up-regulated cell ingrowth and osteogenic
scaffolds showed supportive performance when seeding osteoblastic differentiation was obtained for PDA/collagen/PLA scaffolds. Jang
cells on them. Liu et al. produced PLGA scaffolds with varied macro- et al. fabricated PCL scaffolds via FDM and further adsorbed with BMP-
porous architecture through FDM [55]. By tuning the nozzle inner 2 and umbilical cord serum, which were finally blocked by alginate
diameter, extrusion temperature, pressure, layer thickness and printing layer crosslinked by CaCl2 [61]. The dual delivery scaffolds induced
angles, PLGA scaffolds with pre-designed architecture, height, pore size significantly higher level of osteogenic differentiation and cell miner-
and porosity were obtained. The scaffolds with varied macroscopic alization in vitro and new bone formation in vivo. However, whether the
structure had a compressive strength of 15–23 MPa, which decreased crosslinked alginate layer could reduce the burst release of bio-agents
with increasing scaffold porosity. As PLGA is biodegradable, the pH, into the outer environment was not discussed, which is highly im-
weight remaining and compressive strength of 3D printed PLGA scaf- portant to provide sustained induction for osteogenesis. Ritz et al. de-
folds showed varying degrees of decrease with increasing incubation veloped PLA scaffolds through FDM and they were further loaded with
time. The printed PLGA scaffolds showed favorable cell responses, collagen I and stromal-derived factor-1 (SDF-1) [62]. Both cage- and
however, due to the lack of micropore on scaffold struts, only limited disc-like scaffolds were produced and PLA cages loaded with SDF-1
cells can be observed on scaffold surface after a period of culture. collagen displayed a steady SDF-1 release, supported cell growth of
Mohseni et al. conducted independent evaluation of medical-grade endothelial cells and induced neo-vessel formation in vivo, demon-
bioresorbable filaments for making tissue engineered constructs via strating the potential for PLA scaffolds in bone tissue engineering.
FDM [56]. Raw materials including Dioxaprene® 100 M (DIO), Capro-
preneTM 100 M (CAP), Lactoprene® 100 M (LAC) and Max-Prene®955 5.3. 3D printing of synthetic/natural polymer bone tissue engineering
(MAX) were printed into scaffolds with varied pore size/porosity and scaffolds at room/low temperature
showed distinct mechanical properties according to varied porosity and
matrix type. As these scaffolds had varied hydrophilicity and degrad- Apart from FDM, SLS, etc., extrusion-based 3D printing at room
ability, accelerated degradation and eroded morphology were observed temperature or in a cryogenic environment is considered an advanced
for MAX and DIO scaffolds, whereas LAC and CAP scaffolds showed a 3D printing technique to produce synthetic/natural polymer bone tissue
medium and slow degradation with a more contact morphology, re- engineering scaffolds with capabilities of in situ delivery of biomole-
spectively. Although customized scaffolds can be produced via FDM to cules. By dissolving synthetic polyesters in a variety of organic solvents
repair bone defects, these scaffolds lack of bioactivity to elicit favorable such as 1,4-dioxane, dimethyl sulfoxide (DMSO), etc., printing inks can
cell responses, hence delaying the bone regeneration. be formed and they are capable of loading a large amount of bio-agents
including bioceramic particles, drugs and biomolecules. By tuning the
5.2. Post-treatment of polyester scaffolds 3D printed at high temperature processing parameters, multi-delivery bone tissue engineering scaffolds
could be printed and stabilized. Kim et al. formulated alendronate
Although biodegradable polymeric scaffolds fabricated through (ALN) incorporated PCL/DMSO solution and produced ALN/PCL scaf-
FDM can be used as bone tissue engineering scaffolds, shortcoming such folds through an extrusion-based 3D printing [63]. Sustained ALN re-
as smooth strut surface which hinders effective cell attachment still lease over 4 weeks can be obtained from the ALN/PCL scaffolds which
exists and improvement should be done. By blending graphene with showed no toxicity against MG63 cells. The ALN/PCL scaffolds en-
PCL at a high temperature, Wang et al. fabricated graphene/PCL scaf- hanced the osteoblast activity and mineralization. Moreover, the im-
folds via FDM, in order to enhance the surface hydrophilicity and cell plantation of ALN/PCL scaffolds in a rat tibial defect model greatly
attachment [57]. The addition of pristine graphene was found to have a promoted bone formation compared to pure PCL scaffold. In our study,
positive impact on cell viability and proliferation. Kosik-Koziol et al. we employed P(DLLA-TMC), a thermo-responsive shape memory
conducted surface modification on 3D printed PCL constructs by dip- polymer, as polymer matrix to print rhBMP-2 loaded P(DLLA-TMC)
ping 3D printed PCL scaffolds into acetone and/or NaOH with the as- scaffolds [64] in which rhBMP-2 containing water phase was blended
sistance of ultra-sonication [58]. Much rougher strut surface with a with P(DLLA-TMC)/DCM to form water-in-oil emulsion inks. The scaf-
lowered water contact angle could be obtained. The adsorption rate of fold had excellent compressive strength at room temperature and be-
osteogenic biomolecules such as BMP-2 was significantly improved, came soft at 37 °C, showing great potential to induce bone regeneration
although the mechanical properties were slightly deceased. The density through minimally invasive implantation. Yang et al. further employed
of human MSCs as well as improved osteogenic differentiation was polyester-based water-in-oil Pickering emulsions (silicon dioxide

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nanoparticles as emulsifiers) as inks to produce hierarchical macro- agglomerations of β-TCP were observed, the β-TCP reinforcement im-
porous scaffolds using 3D printing at room temperature [65]. The vo- proved mechanical and hydrophilic behavior in comparison with PCL
lume ratio between PLLA/PCL/DCM and water phase was very low (30: scaffolds. Goncalves et al. reported the construction of composite
70), enabling the production of scaffolds with homogeneous micropores scaffolds comprising of nano HA, carbon nanotubes and PCL matrix via
(20 μm) on struts and very high overall porosity (98.3%). The scaffolds FDM, aiming at bringing together the properties of all components into
also showed favorable capability for improved cell adhesion and pro- a unique material for bone tissue engineering [71]. The 3D printed
liferation, indicating great potential for inducing bone tissue re- composite scaffolds with an interconnected network of square pores
generation. (450–700 μm) and scaffolds containing 2 wt% carbon nanotubes ex-
hibited the best balance between mechanical strength and electrical
5.4. 3D printing of synthetic/natural polymer bone tissue engineering conductivity, showing a compressive strength of 4 MPa, which is
scaffolds via electrostatic field-assisted micro -extrusion comparable with the cancellous bone. The composites induced typical
HA deposition, showing excellent bioactivity and capability for cell
Electrostatic field-assisted micro -extrusion, also known as near- adhesion and spreading. Nyberg et al. fabricated TCP/PCL, HA/PCL,
field electrospinning direct-write is another important 3D printing Bio-Oss/PCL and decellularized bone matrix (dBM)/PCL scaffolds
technique to produce tissue engineering scaffolds with micro- or na- through FDM [72]. The incorporation of mineral particles did not sig-
nofeatures. Ristovski et al. employed direct write near-field melt elec- nificantly decrease the compressive modulus of the graft, which was on
trospinning to produce ordered scaffolds with 200 layers [66]. Pat- the order of 260 MPa for solid blocks and 32–83 MPa for porous scaf-
terned microstructure was obtained (fiber spacing 1 mm and diameter folds. Gene expression of collagen I and osteocalcin was 10-fold greater
40 μm) and successful cell attachment and homogenenous cell dis- than PCL in Bio-Oss/PCL and dBM/PCL when adipose-derived stromal/
tribution in scaffolds was observed, demonstrating the feasibility of stem cells were cultured on scaffolds in vitro, suggesting that Bio-Oss/
using electrostatic control for fabrication of scaffolds with regular mi- PCL and dBM/PCL hybrid materials were advantageous for bone
crostructure, which was desirable for tissue engineering. He et al. also healing applications over HA/PCL or TCP/PCL blends. Park et al. fur-
produced a layer-structured HA/PCL scaffold with uniform pore sizes ther investigated the use of β-TCP/PCL composites with applied me-
which was suitable for 3D cell culture by near-field electrospinning chanical stimulation as scaffold for bone tissue engineering [73]. After
[67]. The results showed that the scaffold had an average pore size of blending ball-milled PCL with β-TCP particles, β-TCP/PCL composite
167 μm, which can be tuned based on the required application; the powders were subjected to FDM, forming bone tissue engineering
degradation rate was controllable depending on the ratio of PCL to HA. scaffolds with a regular latticed macroscopic morphology. The scaffolds
The scaffolds showed no toxicity and MC3T3-E1 cells could effectively were mechanically comparable to human trabecular bone and desirable
attach, proliferate, and differentiate in the 3D skeleton of the scaffolds. cell responses were obtained by seeding MSCs on PCL/TCP scaffolds
Qu et al. constructed microscale biomimetic nano HA/PCL composite with a TCP content of 30%. With mechanical stimulation, expression of
scaffolds for bone tissue engineering via near-field electrospinning as osteogenic markers was lower on samples with a TCP content of 10 wt%
well [68]. The scaffolds had a mostly regular lattice structure with a than without TCP, whereas scaffolds with a TCP content of 30 wt%
pore size of 50–100 μm and the strands had a diameter of 10 μm exhibited significantly higher expression of those markers than the
MC3T3-E1 cells could attach to the fiber surface and proliferate along other samples, suggesting that mechanical stimulation interacted clo-
with culture time. These scaffolds could potentially be used to regulate sely with the addition of TCP, in which TCP with a mass ratio of 30%
cellular microenvironment in multi-scale and multi-material levels for was particularly useful as a bone tissue scaffold when accompanied by
improved bone tissue regeneration. Kim et al. employed electro- mechanical stimulation. Oladapo et al. used carbonatite HA/PLA
hydrodynamic printing, which is principally the same to the near-field composites as raw materials to produced biomimetic composite bone
direct write electrospinning, to produce 3D microfibrous scaffolds with tissue engineering scaffolds through FDM [74]. The printed scaffolds
very high loading level of TCP [69]. The fabricated ceramic structure had a concentric cylinder structure which could closely mimic the
consisted of layer-by-layered struts entangled with PCL microfibers and macroscopic architecture of the segmented long bone. The incorpora-
the bioceramic phase. Different from other near-field electrospinning- tion of carbonatite HA in PLA matrix improved the bioactivity of the
based scaffolds which had a regular latticed microfibrous structure whole scaffolds, hence improving the bone regeneration ability. Simi-
(strut fiber: 1–20 μm, strut fiber distance: 20–100 μm), the struts in this larly, Neumann et al. 3D printed CaCO3/PCL biocomposite scaffolds for
study had a diameter of 500 μm, showing a rough microporous struc- hard tissue regeneration through FDM [75]. CaCO3 crystals were found
ture in which numerous curly PCL microfiber and TCP particles can be to be uniformly distributed in the PCL matrix. Compared to the CaCO3/
observed. Various processing conditions (such as applied electric field, PCL composite materials with a dense structure, which were produced
flow rate, nozzle size, and weight fraction of the bioceramic) were through mould casting, the 3D printed scaffolds had a lowered com-
manipulated to obtain an optimal processing window. Several physical pressive strength (16 vs. 35 MPa) and Young's modulus (160 vs.
and cellular activities using preosteoblasts helped confirm that the 600 MPa), which was still 5-fold higher than that of human cancellous
newly designed bioceramic scaffold demonstrated significantly high bone. As CaCO3 had a very quick dissociation rate in aqueous en-
metabolic activity and mineralization. vironments, 75–95% weight decrease was obtained in PBS-lipase solu-
tion, whereas obvious mineral deposition on the scaffold surface could
6. 3D printing of inorganic agent/polymer composite as bone be observed. Neufurth et al. blended Ca–P microparticles with PCL
tissue engineering scaffolds microparticles with a ratio of 2:1 and transferred the mixed powders
into the printing head heated to 100 °C to conduct 3D printing using a
6.1. 3D printing of bioceramic/polymer composite as bone tissue FDM-like 3D printer [76]. The addition of Ca–P microparticles into PCL
engineering scaffolds matrix not only significantly improved the mechanical properties but
also enhanced the biological performance by showing much more live
In order to improve the osteoconductivity of printed scaffolds, ad- osteoblastic cells on the surface of scaffolds. Shim et al. immobilized
ditives such as bioceramics have been directly blended with polyesters BCP nanoparticles on the surface of 3D printed scaffolds [77]. After
to form composites to produce bioactive scaffolds through FDM. Davila treating PCL scaffolds with L-lysine, aminated-PCL scaffolds were ob-
et al. produced β-TCP/PCL composite scaffolds through a mini-screw tained. BCP nanoparticles were modified with heparin-dopamine (Hep-
extrusion-based 3D printing [70]. The β-TCP acted as nucleating DOPA) to get Hep-BCP nanoparticles.Then, BCP-immobilized PCL (BCP-
agents, allowing a better organization of polymer chains, hence in- PCL) scaffolds were prepared by immobilization of Hep-BCP nano-
creased the crystallinity of the polymer matrix. Although some particles on the surface of aminated-PCL scaffolds. In vitro and in vivo

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results showed that BCP-PCL scaffolds significantly enhanced osteo- differentiation in vitro. Moreover, the biological activity of rhBMP-2
genic markers such as ALP activity, mineralization and osteogenic gene loaded in emulsion inks and scaffolds was well-preserved. We further
expression and markedly increased new bone formation and miner- incorporated graphene oxide (GO) and osteogenic peptide into water/
alized bone tissues in tibial defects, compared to unmodified PCL and TCP/PLGA/DCM inks and printed peptide/GO/TCP/PLGA scaffolds
BCP-mixed PCL scaffolds, demonstrating that the immoblization of BCP [83]. Likewise, scaffolds had a hierarchically porous structure and were
nanoparticles to PCL scaffold surface are promising templates for bone mechanically stronger than human cancellous bone. The addition of GO
tissue regeneration. made the release of peptide more sustained and improved cellular re-
sponses. Both greatly improved in vitro osteogenic differentiation of rat
6.2. Functionalization of 3D printed bioceramic/polymer composite MSCs and in vivo cranial bone regeneration in rats were achieved,
scaffolds suggesting our cryogenic 3D printed scaffolds were suitable for indu-
cing customized bone tissue regeneration. Moreover, emulsion can be
Surface functionalization of composite bone tissue engineering considered as a promising template to tune the microstructure and
scaffolds is an useful route to improve the biological performance of protect biomolecules.
bioceramic/polymer composite scaffolds. Saska et al. produced PHB
bone tissue engineering scaffolds through SLS, followed by post-ad-
sorption of osteogenic growth peptide [78]. The scaffolds had a regular 7. Combinatory use of 3D printing and other fabrication
latticed 3D pattern, in which the struts had a rough surface which was techniques to make bone tissue engineering scaffolds
favorable for peptide adsorption. Duan et al. also employed SLS to
produce Ca–P/PHBV scaffolds and further grafted scaffold surface with To date, to design and fabricate bone tissue engineering scaffolds
heparin for the conjugation of rhBMP-2, in order to realize a sustained with multiple components and functions, the use of a single 3D printing
rhBMP-2 release. Both in vitro and in vivo results demonstrated that the technique might be insufficient. Therefore, combining 3D printing and
rhBMP-2 conjugated Ca–P/PHBV scaffolds could greatly improve os- other fabrication techniques in a rational way could lead to successful
teogenesis [79]. However, post-functionlization only allows limited production of bone tissue engineering scaffolds with a sophisticated
loading amount of agents and relatively fast release rate, resulting in composition and structure, in order to better mimic native bone tissue
insufficient bone forming activity. To increase the loading content of for inducing improved bone regeneration. Kankala et al. produced mi-
biologically active agents (i.e., osteogenic agents), elongate the release crofibrous porous scaffold through 3D printing, assisted by a hybrid
duration and preserve the biological activity during 3D printing pro- approach, in which PLGA scaffolds were first 3D printed via FDM,
cess, advanced 3D printing techniques such as cryogenic 3D printing followed by the immersion of PLGA scaffolds in gelatin and nano-HA
has been used, to produce inorganic/polymer composite bone tissue solutions sequentially, hence forming gelatin/nano-HA/PLGA scaffolds
engineering scaffolds loaded with biomolecules. Lee and Kim mixed α- for bone tissue engineering [84]. These scaffolds with proper de-
TCP (80 v/v%) with collagen solution (4 wt%) and plate-rich plasma gradation, excellent mechanical properties and good biocompatibility
(PRP, 1 mg/mL) to form PRP/collagen/TCP printing inks, which were enabled improved attachment, proliferation and osteogenic differ-
then subjected to extrusion-based 3D printing under a cryogenic en- entiation of MC3T3-E1 cells. So far, the main limitation of cell seeding
vironment at a temperature of −16 to −18 °C [80]. A multi-layered in conventional tissue engineering is inhomogenous cell distribution in
mesh structure was printed, which was then freeze dried for 12 h, scaffolds and poor cell viability in the middle of the scaffold due to
followed by immersion in tannic acid solution to allow the crosslinking limited diffusion of oxygen and nutrients and insufficient vasculariza-
of collagen and immersion in PBS for the hydrolysis of α-TCP, respec- tion. Therefore, the uniform loading of cells in scaffold with high cell
tively. The printed scaffolds had a macro-micro multiscale hier- viability is highly favorable. Guduric et al. used PLA/chlorofrom solu-
archically porous structure. The crosslinking of collagen with tannic tion as printing inks to produce PLA membranes using an extrusion-
acid could increase the mechanical properties, but still showing a lim- based 3D printer [85]. Afterwards, layer-by-layer bioassembly of cel-
ited compressive strength and modulus, which were only 1/10 to 1/5 of lularized (endothelial progenitor cell, human bone marrow stromal cell
that of human cancellous bone. The PRP/collagen/TCP scaffolds up- or cocultures) porous PLA membranes were achieved for bone tissue
regulated the viability, proliferation as well as osteogenic differentia- engineering. In the 3D assembled scaffolds, cell migration between
tion of cells by showing increased expression level of osteopontin and layers of layer-by-layer constructs and osteogenic differentiation were
cell mineralization. Lai et al. utilized TCP/PLGA/1,4-dioxane composite observed, indicating that layer-by-layer assembly of PLA layers was
solution containing icariin as printing inks to produce bone tissue en- suitable for bone tissue engineering to promote homogenous cell dis-
gineering scaffolds in a low temperature environment [81]. The printed tribution inside the scaffold. Based on FDM, Duan et al. fabricated
scaffolds had a hierarchically porous structure and were mechanically composite scaffolds consisting of 3D printed PCL/HA and bioactive
similar to human cancellous bone. The scaffolds degraded gradually hydrogels for the prevascularization of engineered bone tissue con-
with increasing incubation time and the compressive strength as well as structs [86]. The co-culture of MSCs and human umbilical vein en-
Young's modulus were also decreased. With the incorporation of tra- dothelial cells (HUVEC) promoted in vitro vascularization, cell migra-
ditional Chinese herbal medicine, icariin, improved cell proliferation tion, and sprouting, but did not affect osteogenesis. The photocurable
and osteogenic differentiation were achieved in vitro while the new hydrogels provide an engineered vascular bed and facilitated the for-
bone formation and vascular vessel formation was also up-regulated. In mation of microvessels and vasculature. This strategy provide an al-
our investigation, water-in-oil emulsion printing inks (water/PLLA/ ternative for prevascularization of patient specific constructs in vitro to
DCM) containing rhBMP-2 or Ca–P nanoparticles were formulated [82]. achieve rapid anastomosis in vivo and enhance bone repair. Ahlfeld
By alternatingly printing rhBMP-2/water/PLLA/DCM and water/Ca–P/ et al. designed complex bone tissue engineering scaffolds by combined
PLLA/DCM inks, bicomponent bone tissue engineering scaffolds with 3D plotting of a calcium phosphate cement (CPC) and a growth factor-
spatially balanced osteoconductivity/osteoinductivity were constructed loaded hydrogel [87]. After 3D plotting, two-step post-processing in-
in a cryogenic environment at a temperature of −30 °C and DCM were cluding alignate-gellan gum hydrogel crosslinking and CPC setting was
removed via cryo-drying. The fabricated scaffolds had a hierarchically performed. The optimization of CPC plotting enabled the fabrication of
porous structure, showing latticed scaffold pattern and adjustable mi- highly resolved structures with a strand diameter of 200 μm. Micro-
cropores on struts. Scaffolds were mechanically comparable to human computed tomography revealed a precise strand arrangement and an
cancellous bone and provided sustained release of rhBMP-2 and cal- interconnected pore space within the biphasic scaffold even in swollen
cium ions. The scaffolds improved the human MSC attachment, state of the hydrogel strands.
spreading and proliferation and further enhanced the osteogenic

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8. Outlook Acknowledgement

Over the past twenty years, which are very short time for R & D in This work was supported by Dongguan University of Technology
science and technology, 3D printing has advanced at a phenomenal High-level Talents (Innovation Team) Research Project
pace due to its advantages in producing products with customized (KCYCXPT201603), Youth Innovative Talent Project from the
shape, tailored structure, adjustable composition, etc., and wide ap- Department of Education of Guangdong Province, China
plications of the products by many industries, especially the biomedical (2016KQNCX168), Natural Science Foundation of Guangdong
industry. The initial exploration on 3D printed scaffolds soon yielded Province, China (2018A0303130019).
great excitement in bone tissue engineering fields. The limitations of
conventional 3D printing on making advanced bone tissue engineering References
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